By their nature, organisms contain many complex molecules and molecular assemblies. Some of the most important molecules and assemblies are large and have high aspect ratios (i.e. one axis significantly greater in length than any other). It is known to use an optical apparatus to specifically detect these high aspect ratio molecules. Such an apparatus relies on the way these long molecules interact with polarised light (i.e. light with an electric field established in one direction only).
The phenomenon being exploited in the above apparatus is known as dichroism. The incident light may be either linearly polarised, giving rise to linear dichroism (LD), or circularly polarised, giving rise to circular dichroism (CD). LD is the property exhibited by some molecular structures whereby linearly polarised light is differentially absorbed along two orthogonal axes. CD relates to the difference in absorption of left and right circularly polarised light. A molecule that is capable of selective light absorption is known as a chromophore. Dichroic molecules, i.e. those that exhibit dichroic properties, are a particular type of chromophore. Examples of dichroic materials are certain natural crystals, stretched polymers, and other non-isotropic molecules. Biomolecules contain a wide range of chromophores (including aromatic side chains, nucleotides and peptide backbones).
In order to be able to observe a dichroic effect, it is necessary that the chromophores be aligned, or at least partially aligned, with respect to the incident polarised light beams. This requirement has the advantage of allowing the extraction of data only from aligned molecules in a milieu of unaligned molecules. However, this requirement has, to date, also limited the application of the above technique, primarily, to the study of large molecules with high aspect ratios, since these are easily alignable. A molecule is considered to have a high aspect ratio if one axis is substantially longer than the other. Suitable molecules may be in the shape of a rod, a disc or a cruciform. Depending upon the stiffness of the molecule, an aspect ratio of 100:1 may be sufficient to facilitate alignment but an aspect ratio of greater than 1000:1 is preferable. Some examples of moieties of interest that have been successfully aligned include linear biomolecules in the form of DNA, fibrous proteins and membranes (including membrane proteins) (Marrington R, Small E, Rodger A, Dafforn T R, Addinall S G, “FtsZ fiber bundling is triggered by a conformational change in bound GTP” J Biol Chem 2004; 279(47):48821-48829; Dafforn T R, Rajendra J, Halsall D J, Serpell L C, Rodger A, “Protein fiber linear dichroism for structure determination and kinetics in a low-volume, low-wavelength couvette flow cell” Biophys J 2004; 86(1 Pt 1):404-410; Dafforn T R, Rodger A, “Linear dichroism of biomolecules: which way is up?” Curr Opin Struct Biol 2004; 14(5):541-546; Halsall D J, Rodger A, Dafforn T R, “Linear dichroism for the detection of single base pair mutations” Chem Commun (Camb) 2001 (23):2410-2411).
A particularly convenient method for aligning such molecules is to create a solution including the molecules and then to flow the solution. Due to the elongate nature of the molecules, alignment arises as a result of shear forces generated by the flow, making the sample suitable for exhibiting the effect of linear dichroism.
In a known apparatus, once the molecules of interest have been aligned, linearly polarised light is directed through the solution from a direction substantially perpendicular to the axes of the aligned molecules. Absorption of light occurs within a molecule because, at a particular wavelength, the electric field of radiation urges the electrons in the molecule in a particular direction. When several molecules are similarly aligned, the electrons in each are all characterised by the same preferred net displacement direction. LD is a measure of the difference of absorbance of the incident light between two orthogonal polarisations. Varying the wavelength of the incident light and detecting the light emerging from the sample, allows a spectrum to be obtained which illustrates the absorbance of the sample with respect to wavelength.
An LD spectrum of a molecule provides information on the chromophores that are present including the orientation of the chromophores (and hence molecular conformation) and the orientation of the chromophores with respect to the axes of polarization. This information is important in understanding the structure of the molecule. Note that LD is a measurement of a sample's bulk property. The strength of the absorbance can be used to quantify the number of target molecules that are present in the sample. In addition, since LD is extremely sensitive to changes in alignment, an anomaly in the structure of a molecule may be detected. For example, LD can detect the distortion caused by a single mismatched hydrogen bond in a 1300 bp (base pair) fragment of DNA.
Furthermore, LD is extremely sensitive to the formation of a complex since the binding of an aligned molecule to a second molecule has the following two measurable effects.                1) The shape of the aligning moiety is altered and this results in its alignment also being altered, which leads to a change in the observed LD spectrum.        2) The second molecule itself becomes aligned by virtue of its attachment to the aligned molecule. This leads to the generation of an LD signal for the previously unaligned chromophores of the second molecule. Thus, information on the structure of the complex can be obtained.        
Both of the above effects result in detectable phenomena that can be used to detect the formation of complexes. Not only can structural information be gleamed regarding the nature of the complex but the affinity of the interaction can also be determined.
Unfortunately, most molecules do not have a high aspect ratio and instead have shapes more closely related to spheres, with aspect ratios of less than approximately 5:1. In order to align these molecules it is necessary to link the target molecule to a receptor that itself has a high aspect ratio. This method of alignment has been achieved and has been applied to studies of ligands (e.g. cisplatin) that bind to naturally alignable receptors (e.g. DNA). However, this method is also limited in its application since only those molecules that bind to naturally alignable receptors can be studied.
It is therefore an aim of the present invention to expand the application of dichroic analysis.